Speciation of Chromium(vi) and Chromium(iii) Using Pneumatically Assisted Electrospray Mass Spectrometry ALBIN B. GWIZDALA III†, STEVE K. JOHNSON, SAHANA MOLLAH AND R. S. HOUK* Ames L aboratory—U.S. Department of Energy, Department of Chemistry, Iowa State University, Ames, IA 50011, USA Mass spectra can be obtained from aqueous solutions and oxoanions of the same element under the same spray conditions. containing both Cr3+ and Cr2O72- as negative ions under the same spray conditions.An excess of HCl is used so that Cr3+ sprays as an anionic chloro complex. Moderately energetic EXPERIMENTAL collision conditions produce CrO3- from CrVI and CrOCl2- from CrIII. Detection limits are 100 and 60 ppb for CrIII and Standard solutions were prepared by diluting aliquots from CrVI, respectively. Reasonable calibration curves are provided 1000 ppm aqueous standards (ICP emission standards from by plotting the ratio of the analyte signal to that for 37Cl-. Aldrich Chemicals) with a 50% methanol–50% water solvent. The methanol–water solution was prepared using HPLC grade Keywords: Chromium determination; speciation; electrospray; methanol(Fisher Scientific) and water de-ionized to a resistivity ion spray; mass spectrometry of 18 MV cm( with a Barnsted Nanopure-II system (Newton, MA, USA).ULTREX II ultra pure grade HCl (J. T. Baker) Speciation information is needed as the toxicity and biological was used to bring the final concentration (v/v) of solutions up role of a particular element can vary greatly depending on the to 1% HCl.Approximately 500 ml of solution were drawn into chemical form. At low levels, CrIII is essential for living organ- a 1 ml syringe (Hamilton). Solutions were transported from isms. It is an essential nutrient and is believed to help activate syringe to ion source through a 100 mm id fused silica capillary insulin.1 On the other hand, CrVI can cross cell membranes (Polymicro Technologies) using a syringe pump (74900 Series, and cause skin lesions, lung cancer and other forms of cancer.2 Cole-Parmer Instruments).Chromium has many industrial applications such as dyeing, A Perkin-Elmer SCIEX API 1 mass spectrometer was used tanning and use in the steel industry. Accurate determination (Fig. 1). Typical conditions used for the instrument are summaof each species rather than just the total chromium level is rized in Table 1. Voltages were optimized on a daily basis to important in determining toxicity.maximize the signal for the species of interest. The ‘best’ In the late 1960s, Dole and co-workers3,4 described the voltages varied slightly (±5 V) from day to day; typical values formation of gas-phase ions when a liquid was sprayed out are listed in Table 1. Peak hopping data were collected using of a capillary held at high voltage. Since that time, the eorts a 100 ms dwell time. Spectral scans were collected by adding of Fenn and co-workers5–8 have led to the development of ten consecutive scans together using a 10 ms dwell time.electrospray mass spectrometry (ES-MS). Pneumatically assisted electrospray, also referred to as ion spray, was first RESULTS AND DISCUSSION reported by Bruins et al.9 Ion spray can be considered as a concentric pneumatic nebulizer combined with electrospray. Selection of Collision Conditions These two terms are often used interchangeably. Since the The polarity of the voltage applied to the electrospray needle development of electrospray and ion spray, many applications determines whether positive or negative ions are observed.The have been discovered. Electrospray has found widespread use collision conditions then determine the extent of fragmentation for the determination of the relative molecular mass of large observed. In this experiment, the dierence between the orifice biological molecules.10–12 This technique has also been used plate voltage (VOR, see Fig. 1) and the voltage on the RF-only for the determination of many inorganic species, from ion rods (VRF) has the largest eect on the chromium species clusters to bare metal ions.13–21 Using electrospray mass spectrometry, it is easy to distinguish Cr3+ from Cr2O72- using separate spray conditions.21 A positive voltage is applied to the electrospray needle to spray Cr3+, while a negative voltage produces anions from Cr2O72-. This procedure requires separate runs to observe both cations and anions.The present work describes a possible way to distinguish CrIII and CrVI with the same spray conditions. The cation Cr3+ is sprayed as a negative chloro complex. Horlick and co-workers refer to this as the ‘intermediate’ or ‘counter ion’ mode because collisions during the ion extraction process occur at moderate kinetic energies such that anions remain associated with the metal cation.20 This concept could become a general procedure for distinguishing cations Fig. 1 Schematic diagram of electrospray mass spectrometer including ion source, interface assembly and some ion optics. Representation † Present address: U.S. Silica, Box 187, RT 522 North, Berkeley Springs, WV 25411, USA. is not drawn to scale. Journal of Analytical Atomic Spectrometry, May 1997, Vol. 12 (503–506) 503Table 1 Typical operating conditions 17 ml min-1 Sample flow rate Nebulizer gas Nitrogen Nebulizer gas pressure 280 kPa Curtain gas Nitrogen, ultra pure carrier grade Curtain gas back pressure 550 kPa Curtain gas temperature 60 °C Ionization needle voltage (VISV) -3200 V Interface plate voltage (VIN) -500 V Orifice plate voltage (VOR) -125 V RF only quadrupole voltage -60 V (VRF) Mass analyzer quadrupole -59 V voltage (VR1) CEM detector voltage +2500 V Fig. 3 A plot of signals from the major Cr species, A 52CrO3-, m/z= Operating pressure of 5.333×10-3 Pa 100; B 52CrO335Cl-, m/z=135, observed from a 10 ppm CrVI solution quadrupole chamber while varying the voltage on the orifice plate.Fig. 2 A plot of signals from the major Cr species, A 52CrO235Cl-, m/z=119; B 52CrO35Cl2-, m/z=138; C 52CrO35Cl2- H2O, m/z=156; D 52CrO35Cl2- 2H2O, m/z=174, observed from a 50 ppm CrIII solu- Fig. 4 Mass spectrum of a 50 ppm CrIII solution. The inset is the tion while varying the voltage on the orifice plate. region from m/z=95 to 105. (CrCl4 2H2O)-, or something similar, but such ions could not detected. Ions from the spray flow through the 200 mm diameter be observed even under the ‘softest’ extraction conditions.orifice in the orifice plate, along with nitrogen from the curtain Fig. 3 shows a plot of signal versus orifice voltage (VOR ), gas. As the ions are accelerated through the potential dierence from a 10 ppm CrVI solution. At a VOR-VRF of between 10 (VOR-VRF), they collide with nitrogen molecules in the superand 50 V, the chromium oxochloro species CrO3Cl- (m/z= sonic jet that forms behind the orifice plate.The larger the 135) is observed. At slightly more harsh collision conditions, voltage dierence (VOR-VRF), the more energetic the collision the ion CrO3- (m/z=100) is observed. Using conditions that conditions, which produce more extensive fragmentation. This are favorable for detecting CrOCl2- (m/z=138) from the CrIII voltage dierence is essentially the collision energy for collisionsolution (-125 V on VOR, VOR-VRF#65 V), both CrO3- and induced dissociation in the laboratory frame of reference.CrO3Cl- are observed from CrVI, with CrO3- being the In the present work, the voltage dierence (VOR-VRF) was dominant species. varied by changing VOR only. The ion kinetic energy inside the Thus, a voltage dierence of 65 V between VOR and VRF mass filter is determined by the dierence (VRF-VR1); this produces stable chromium oxo or oxochloro species from both dierence is kept constant so the resolution and peak shapes CrIII and CrVI solutions.These conditions were used to obtain do not change greatly as VOR is altered. all the following results unless otherwise indicated. It should Fig. 2, a plot of signal versus VOR, was generated from a be noted in Fig. 3 that the signal at m/z=100, between -60 V 50 ppm CrIII solution. The plots shown in Fig. 2 are for various and -90 V, is not due to Cr. Scans under these conditions chromium oxochloro species identified in the caption. The ions reveal no Cr isotope pattern in this region.Scans do reveal selected contain 52Cr and 35Cl and are the most abundant species present at both m/z=98 and m/z=100, in a 3 to 1 ones from the various isotope patterns. The main ions under intensity ratio. This would seem to indicate that this species soft conditions (VOR#90 V, VOR-VRF #30 V) still have chrocontains chlorine and is probably due to a background ion. mium as CrIII and intact oxygen ligands. As can be seen in Fig. 2, using a VOR value of -125 V (i.e., VOR-VRF# 65 V) yields a maximum signal for CrOCl2- (m/z=138).Mass Spectra Using -125 V on VOR minimizes the abundance of (CrOCl2 nH2O)- ions. Fig. 4 shows a mass spectrum of a 50 ppm CrIII solution obtained under the optimum conditions (VOR-VRF=65 V) The onset voltage for observation of the hydrated ions is VOR#-80 V. The onset voltages for the analyte species described above. The most abundant chromium oxochloro species are observed at m/z=138, 140 and 142.This set of CrO2Cl- and CrOCl2- are approximately 15 V more negative. This 15 V dierence represents the additional kinetic energy peaks is attributed to CrOCl2- and appears in the appropriate isotope ratios, 95651, for an ion with two chlorine atoms. Less necessary to create CrO2Cl- and CrOCl2- from whatever anion is formed originally. Presumably, this precursor ion is intense peaks can also be observed at m/z=119 and 121; the 504 Journal of Analytical Atomic Spectrometry, May 1997, Vol. 12351 isotope pattern shows they have one chlorine atom. These are m/z=100, CrO3- from CrVI, and m/z=138, CrOCl2- from CrIII. In addition to the peaks associated with the isotope peaks are likely to be CrO2Cl-. The region at m/z=100, shown in the inset of Fig. 4, does not contain ions from Cr3+. patterns from the previously mentioned species, CrO2Cl- from CrIII and CrO3Cl- from CrVI are also observed. A mass spectrum of CrVI at 10 ppm is shown in Fig. 5. Chromium species can be observed in the m/z=100 and m/z= The CrO3- ion that is formed from CrVI actually contains Cr in the oxidation state +5. No Cr2O72- or CrO42- were 135 regions. The intensities of the peaks around m/z=100 agree very well with the known isotope ratio of chromium. detected, even under the softest ion extraction conditions. As pointed out by Stewart and Horlick,21 Cr2O72- is readily The dominant peak at m/z=100 is assigned to 52CrO3-. The set of peaks at m/z=135 and 137 are probably CrO3Cl-.The reduced by methanol, which explains why intact ions from chromate or dichromate were not observed. Nevertheless, the peak observed at m/z=113 in Fig. 5 and another fragment peak at m/z=69 (not shown) are due to contamination of the CrO3- ion is characteristic of CrVI in the original sample, i.e., before the methanol was added to enhance the electrospray instrument with trifluoroacetate (TFA-). The TFA contamination can be seen throughout this study and is a result of process.other experiments on electrospray of organic acids. Cleaning procedures have as yet been unable to remove the TFA Calibration Curves and Detection Limits completely from the instrument. TFA does not interfere with the determination of chromium but does appear in these scans. Calibration curves and detection limits were determined for However, this observation does illustrate one possible problem: both CrIII and CrVI solutions. Calibration curves for CrIII and organic ions that remain intact can cause memory or spectral CrVI are shown in Figs. 7 and 8, respectively. These plots were interferences. generated by taking the ratio of the analyte signal, m/z=138 During this work, when an aqueous CrVI solution was for CrIII or m/z=100 for CrVI , to the signal at m/z=37 due to allowed to stand, a small amount of CrVI was reduced to CrIII. 37Cl- from the aqueous HCl solvent. As described by Agnes The characteristic peaks for CrIII can then be seen from the and Horlick, it is often advantageous in ESMS to ratio the CrVI solution. This is a common problem with inorganic analyte signal of interest to another signal to compensate for speciation.When species are sampled, care should be taken to avoid changing the species present in the initial sample. Sampling and sample preparation work can alter the speciation information and lead to inaccurate measurements. Fig. 6 shows a mass spectrum of a mixture containing 50 ppm CrIII and 10 ppm CrVI.The dominant peaks observed Fig. 7 Calibration curves for CrIII solutions: (a) wide range concentration plot, including non-linear region; and (b) a plot of the lower concentration region of (a). Fig. 5 Mass spectrum of a 10 ppm CrVI solution. Fig. 8 Calibration curves for CrVI solutions: (a) wide range concen- Fig. 6 Mass spectrum of a mixture containing 50 ppm CrIII and tration plot, including non-linear region; and (b) a plot of the lower concentration region of (a). 10 ppm CrVI. Journal of Analytical Atomic Spectrometry, May 1997, Vol. 12 505Table 2 Detection limits REFERENCES 1 da Silva, J., and Williams, R., T he Biological Chemistry of the Species Relative/ppb Absolute/pg Elements, the Inorganic Chemistry of L ife, Clarendon Press, CrIII 100 3 Oxford, NY, 1991, p. 541. CrVI 60 1.5 2 Ottaway, J. M., and Fell, G. S., Pure Appl. Chem., 1986, 58, 1701. 3 Dole, M., Mach, L. L., Hines, R. L., Mobley, R. C., Ferguson, L.P., and Alice, M. B., J. Chem. Phys., 1968, 49, 2240. 4 Mach, L. L., Kralik, P., Rheude, A., and Dole, M., J. Chem. Phys., the variation of electrospray signal with the total ionic com- 1970, 52, 4977. position of the sample.20 Fairly linear calibration curves (cor- 5 Whitehouse, C. M., Dreyer, R. N., Yamashita, M., and Fenn, J. B., Anal. Chem., 1985, 57, 675. relation coecients of 0.989 for CrIII and 0.992 for CrVI, 6 Meng, C. K., Mann, M., and Fenn, J. B., Z.Phys. D, 1988, 10, 361. respectively) were observed at low analyte concentrations, i.e., 7 Wong, S. F., Meng, C. K., and Fenn, J. B., J. Phys. Chem., 1988, up to 1 ppm for CrIII and 10 ppm for CrVI. The calibration 92, 546. curves roll over at higher analyte concentrations. Detection 8 Mann, M., Meng, C. K., and Fenn, J. B., Anal. Chem., 1989, limits are shown in Table 2. These values represent the solution 61, 1702. concentration necessary to produce a net signal equivalent to 9 Bruins, A.P., Covey, T. R., and Henion, J. D., Anal. Chem., 1987, 59, 2642. three times the standard deviation of background during 10 Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F., and Whitehouse, single-ion monitoring for the dwell time used (0.1 s). The C. M., Science, 1989, 246, 64. detection limits are 60–100 ppb (relative) or 1.5–3 pg (absolute). 11 Smith, R. D., Loo, J. A., Edmonds, C. G., Barinaga, C. J., and Udseth, H. R., Anal. Chem., 1990, 62, 882. 12 Smith, R.D., Loo, R. J., Ogorzalek-Loo, R. R., Busman, M., and CONCLUSION Udseth, H. R., Mass Spectrom. Rev., 1991, 10, 359. 13 Siu, K. W. M., Gardner, G. J., and Berman, S. S., Rapid Commun. The concept described in this paper could become a general Mass Spectrom., 1988, 2, 201. procedure for distinguishing cations and oxoanions of a par- 14 Cheng, Z. L., Siu, K. W. M., Guevremont, R., and Berman, S. S., ticular element under the same spray conditions, in cases where Org. Mass Spectrom., 1992, 27, 1370. both forms remain stable in the same solution. Diculties 15 Blades, A. T., Jayaweera, P., Ikonomu, M. G., and Kebarle, P., include high background, mediocre detection limits and prob- Int. J. Mass Spectrom. Ion Processes, 1990, 101, 325. able spectral interferences from either organic or inorganic 16 Agnes, G. R., and Horlick, G., Appl. Spectrosc., 1992, 46, 401. 17 Stewart, I. I., and Horlick, G., T rends Anal. Chem., 1996, 15, 80. anions. These last problems could be alleviated to a large 18 Colton, R., D’Agostino, A., and Traeger, J. C., Mass Spectrom. extent by the use of tandem mass spectrometry, which should Rev., 1995, 14, 79. eliminate much of the background. 19 Corr, J. J., and Anacleto, J. F., Anal. Chem., 1996, 68, 2155. 20 Agnes, G. R., and Horlick, G., Appl. Spectrosc., 1994, 48, 649, 655. Ames Laboratory is operated for the U.S. Department of 21 Stewart, I. I., and Horlick, G. J. Anal. Atom. Spectrom., 1997, Energy by Iowa State University under Contract Number 11, 1203. W-7405-Eng-82. This research was supported by the Oce of Technology Development, Environmental Management Paper 6/06413B Program (EM-50). Received September 17, 1996 Accepted January 2, 1997 506 Journal of Analytical Atomic Spectrometry, May 1997, Vol. 12